We studied mechanisms for T cell recognition of antigens in association with major histocompatibility complex (MHC)-encoded molecules, and applications to the design of synthetic vaccines for AIDS and cancer. We have been characterizing the helper and cytotoxic T lymphocyte (CTL) responses to HIV envelope and reverse transcriptase, mapping the key epitopes, and defining the role of individual residues in these epitopes to be able to modify the structures to make more potent immunogens as vaccines. Currently, we are developing new approaches in mouse models to develop second generation vaccine constructs. We have shown proof of principle that we can modify the sequence of a helper epitope of HIV to make it more immunogenic and also much more potent, when coupled to a CTL epitope, in eliciting CTL and protecting against viral infection. We have applied this "epitope enhancement" approach to conserved HIV helper and CTL epitopes from env, gag, and pol, presented by human class II and class I HLA molecules, as well as to hepatitis C virus (HCV) epitopes and cancer antigen epitopes presented by human HLA-A2.1 (see below). We have developed an enhanced HIV reverse transcriptase epitope which is the subject of a clnical trial to be carried out with Dr. Robert Yarchoan, HAMB, CCR, NCI, and we have characterized human responses to an envelope helper epitope, which we have also enhanced. We have discovered ways of increasing CTL, helper, and antibody responses and steering them toward desired phenotypes, such as Th1 or Th2 or particular antibody isotypes, by incorporating cytokines into the emulsion adjuvant with the antigen. We have even further enhanced combinations of cytokines by a push-pull approach in which a potent combination of cytokine and costimulatory molecule is complemented by blocking a suppressive pathway with an inhibitor of IL-13, optimizing the vaccine-induced CTL response and protection. We have previously shown that high avidity CTL specific for HIV-1 envelope peptide are much more effective at clearing a recombinant vaccinia virus expressing HIV gp160 from SCID mice than are low avidity CTL specific for the same peptide-MHC complex, and have worked out two complementary mechanisms involving the ability of high avidity CTL to kill cells earlier in virus infection before viral progeny are produced, and to lyse targets more quickly. We recently found that immunodominance depends more on CTL avidity than on the level of the epitope expressed. We have finally developed methods to preferentially elicit higher avidity CTL with a vaccine, by using costimulatory molecules to allow a response at lower antigen dose, and by incorporating IL-15 in the vaccine construct, selecting for cells that have higher IL-15Ralpha, and upregulating CD8. Indeed, we found that IL-15 incorporated in a vaccine induces CTL of a different character that are longer-lived memory cells, with higher levels of IL-15Ralpha, greater responsiveness to IL-15 in vitro and greater homeostatic proliferation in vivo, and higher avidity for antigen. We have shown that high avidity CTL express higher levels of IL-15 receptor alpha chain (IL-15Ralpha) and that this allows these cells to undergo more homeostatic proliferation in response to endogenous levels of IL-15 and survive, whereas low avidity CTL with lower levels of this receptor die out over time, providing an explanation for the long-standing enigma of T cell avidity maturation. We also found that IL-15 upregulates expression of the CD8 coreceptor. Thus, IL-15 mediates two mechanisms of T cell avidity maturation, selection at the population level and instruction at the single cell level. These mechanisms can explain the longstanding enigma of T cell avidity maturation. Use of this cytokine in vaccines should allow induction of longer-lived, higher avidity CTL that are more efficacious.